Salmonella pathogenesis is characterized by the invasion and penetration of the intestinal epithelial barrier. Entry into host host cells requires dramatic reorganization of the actin cytoskeleton, which in non-polarized cells is mediated by members of the Rho family of GTPases. However, the apical actin cytoskeleton of epithelial cells is highly specialized, and we have found that a member of the ADP-ribosylation factor (ARF) family of GTPases, ARF6, may also mediate Salmonella internalization in polarized epithelial cells. In Specific Aim 1, we will examine the specific roles of Rho GTPases and ARF6 in Salmonella internalization and signaling at the apical plasma membrane. Once inside the host cell, Salmonella reside within vacuolar bodies derived from host cell membranes that support their intracellular replication, transit to the basolateral plasma membrane and subsequent release into the lamina propria. These vacuoles are essentially large and specialized endosomes diverted by the bacteria from normal membrane trafficking pathways, and their composition changes with time as the bacteria migrate from the apical to basolateral pole of the cell. Using an MDCK cell model, in which epithelial cells are cultured on permeable filter supports, we have shown that the emergence of bacteria from the basolateral pole of the cell is a vectorial, non-random process, suggesting that Salmonella recruit components of the host cell targeting machinery to direct their transport to and fusion with the basolateral membrane. In support of this hypothesis, we have identified one member of the rab family of small GTPases, rab5, that is present on Salmonella-containing vacuoles at an early stage of biogenesis. In Specific Aim 2, we will determine the function of rab5 in vacuolar maturation, and identify other rabs that may participate in this process. It is widely accepted that IgA protects mucosal surfaces from bacterial invasion by preventing the adhesion of bacteria and their products to the epithelial cell surface. However, it has also been hypothesized that IgA may act intracellularly, to inhibit the replication of and enhance the clearance of intracellular pathogens from the cell. In Specific Aim 3, we will determine whether intracellular IgA interacts with transmigrating Salmonella, and whether such interaction is sufficient to perturb vacuolar biogenesis or transport. The results of these studies will provide significant insight into the mechanisms of Salmonella pathogenesis within the intestinal epithelium.